Local chemical sympathectomy of rat bone marrow and its effect on marrow cell composition.

Existing experimental studies of the effect of sympathetic nerve fibers on bone marrow cells are based on the systemic administration of neurotoxic 6-hydroxydopamine. The method of global chemical sympathectomy has some serious disadvantages and could lead to questionable results. We describe a new method of local chemical sympathectomy of rat femoral bone marrow using guanethidine (Ismelin) delivery using an osmotic mini pump. Local guanethidine treatment for 14days led to complete elimination of sympathetic fibers in femoral bone marrow in contrast to bone marrow of contralateral or naïve femurs. Ablation of sympathetic fibers was associated with a loss of rat endothelial cell marker (RECA) indicating immunophenotype changes in blood vessel endothelial cells, but no significant effect of guanethidine was found on the survival of endothelial cells and mesenchymal stem cells in vitro. Moreover, local guanethidine treatment also elicited a significant reduction of Nestin+/SDF1+ mesenchymal stem cells and c-Kit+/CD90+ hematopoietic stem cells in femoral bone marrow. Tissue-specific chemical sympathectomy of rat bone marrow by guanethidine overcomes some of the drawbacks of systemic administration of neurotoxic compounds like 6-hydroxydopamine and delivers unequivocal evidence on the effects of sympathetic innervation on the cell content of bone marrow.

Quantification of Pyrenophora teres in infected barley leaves using real-time PCR.

Net blotch is a barley foliar disease caused by two forms of Pyrenophora teres: Pyrenophora teres f. teres (PTT) and Pyrenophora teres f. maculata (PTM). To monitor and quantify their occurrence during the growing season, diagnostic system based on real-time PCR was developed. TaqMan MGB (Minor Groove Binder) primers and probes were designed that showed high specificity for each of the two forms of P. teres. As a host plant internal standard, TaqMan MGB primers and probe based on RacB gene sequence were designed. The method was optimised on pure fungal DNA and on plasmid standard dilutions. Quantification was accomplished by comparing Ct values of unknown samples with those obtained from plasmid standard dilutions. The assay detects down to five gene copies per reaction. It is able to produce reliable quantitative data over a range of six orders of magnitude. The developed assay was used to differentiate and quantify both forms of P. teres in infected barley leaves. Correlation R(2)=0.52 was obtained between the Ct values and size of symptoms areas in early stage of infection. Application of the TaqMan MGB technology to leaf samples collected in 20 barley varieties in the region Kromeriz during the growing season of 2003 and 2004 revealed that P. teres f. teres predominated in these 2 years. The developed method is an important tool to quantify and monitor the dynamics of the two forms of P. teres during the growing season.

Coding region single nucleotide polymorphism in the barley low-pI, alpha-amylase gene Amy32b.

Barley alpha-amylase variability influences the quality of barley grain in the brewing, feed and food industries. alpha-Amylase proteins are encoded by multigene families in cereals, and this study focused on the barley Amy32b gene. We identified coding region single nucleotide polymorphism (cSNP) and insertion/deletion variation in DNA sequences, which resulted in amino acid substitution and stop codon formation, respectively. The substitution affected the beta1 strand in domain C, whereas the stop codon removed the beta5 strand. Possible effects of these changes on the protein are discussed. A cSNP in the coding region of the Amy32b gene was used as a specific marker to map Amy32b loci on chromosome 7H.

Cellular metabolism in lymphocytes of a novel thioether-phospholipid-AZT conjugate with anti-HIV-1 activity.

We previously synthesized a thioetherphospholipid-AZT conjugate (3'-azido-3'-deoxy-5'-(1-hexadecylthio-2-methoxypropyl)-phosphothymidine, CP-102) with potent anti-HIV-1 activity and significant reduction in cell cytotoxicity compared to AZT alone. To study the cellular metabolism of the conjugate compound we synthesized a double-tritium-labeled thioetherphospholipid-AZT conjugate (3'-azido-3'-deoxy-5'-(1-[9,10-3H]-S-octadecylthio-2-O-methoxypropyl)-phosphothymidine-[methyl-3H], [3H]CP-102). The intracellular radioactive metabolic products of [3H]CP-102 treated human lymphoblastoid CEM-SS cells were analyzed by HPLC and thin-layer chromatography. Results of this investigation provide evidence that a putative intracellular lipid cleavage enzyme metabolizes [3H]CP-102 to form a thioetherdiglyceride compound that migrates with an authentic 1-S-octadecyl-2-O-methyl-thioglycerol standard on TLC. The thioetherdiglyceride metabolite did not react with the ninhydrin reagent indicating it did not contain a primary amine such as that found on serine or ethanolamine containing phospholipids. Also, the product did not contain a phosphatidic acid group based on migration characteristics in the TLC plate. The other major hydrophilic metabolite was 3'-azido-3'-deoxythymidine-[methyl-3H]-monophosphate (AZT-MP) with lesser amounts of AZT, AZT-DP and AZT-TP. In summary, the best interpretation of these data is that the thioetherphospholipid-AZT conjugate, [3H]CP-102, is cleaved by a putative intracellular lipid cleavage enzyme to release a thioetherdiglyceride compound and AZT-MP. The resulting AZT-MP was either dephosphorylated to AZT or sequentially phosphorylated to AZT-DP and, ultimately, to AZT-TP, the known inhibitory metabolite against HIV-1 reverse transcriptase. Phospholipid-nucleoside conjugates may provide a unique approach for developing anti-HIV-1 prodrugs that do not have a strict requirement for a nucleoside kinase for initial activation of the prodrug to an antiviral form.

Structure-activity relationships for enhancement of paracellular permeability by 2-alkoxy-3-alkylamidopropylphosphocholines across Caco-2 cell monolayers.

The oral route is the preferred route of delivery for a large number of drug molecules. However, the intestinal epithelium presents a formidable barrier for delivery of drugs into systemic circulation. Phospholipids are among compounds that enhance the absorption of drugs across the intestinal epithelium. In this paper, we describe structure-activity relationships for phospholipid derivatives as enhancers of paracellular permeability across Caco-2 cell monolayers. In a series of 2-alkoxy-3-alkylamidopropylphosphocholine derivatives, compounds with a long chain at C-3 (R3) and short chain at C-2 (R2) were potent in causing a decrease in transepithelial electrical resistance (TEER) and an increase in mannitol transport, but also showed significant cytotoxicity. Compounds with 9-11 carbons at C-3 and 6-10 carbons at C-2 provided good separation (up to 2.7-fold) between activity and cytotoxicity. Notably, a good correlation (r2 = 0.93) was observed between EC(50) (TEER) [concentration that caused a drop in TEER to 50% of its control (untreated) value] and EC10x (mannitol) [concentration that caused 10-fold increase in mannitol transport over the control (untreated) value], confirming that a decrease in TEER is associated with enhanced permeability of the hydrophilic compounds across Caco-2 cell monolayers. Compounds with medium to long carbon chains at C-2 and C-3, and the total carbons in the alkyl chains > 20, showed poor activity and no cytotoxicity.

Amplified Assay for Specific Dual-Labeled DNA Using the Coagulation Cascade (EDNA-ELCA).

Detection of the products of the PCR reaction using nonisotopically labeled DNA molecules containing biotin, fluorescein, or digoxigenin has become a popular method for identification of specific products of polymerase chain reaction (PCR) (1,3). These labeled molecules are prepared either by PCR synthesis in the presence of labeled deoxyuridine triphosphate (1,3) or by hybridization of labeled probes to the unlabeled PCR product (1,2). Detection is then in a format very similar to enzyme-linked immunosorbent assays (ELISA) using similarly labeled antigens and antibodies, i.e., by capture on the receptor for one ligand (streptavidin or antibody) and using the other ligand for detection.

Design, synthesis, and antiviral evaluations of 1-(substituted benzyl)-2-substituted-5,6-dichlorobenzimidazoles as nonnucleoside analogues of 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole.

We have recently reported that certain ribosylated polyhalogenated benzimidazoles are potent and selective inhibitors of HCMV replication at noncytotoxic concentrations. To extend the structure-activity relationship beyond these first-generation compounds, we alkylated 5,6-dichloro-2-substituted-benzimidazoles with either a series of substituted benzyl halides or (2-bromoethyl)benzene to obtain five series of nonnucleoside analogues. Evaluation of these compounds for activity against herpes viruses revealed that the new compounds were less active than the benzimidazole ribonucleosides against human cytomegalovirus (HCMV) and inactive against herpes simplex virus type 1 (HSV-1). However, as part of our broader antiviral testing, we found that some of these compounds were active against HIV. Comparisons of the biological data revealed that a chloro or bromo group was required at the 2-position for the best separation of activity against HIV and cytotoxicity. Evaluation of the most active compounds against drug-resistant HIV suggested that they act by a mechanism other than inhibition of reverse transcriptase.

In vitro evaluation and characterization of newly designed alkylamidophospholipid analogues as anti-human immunodeficiency virus type 1 agents.

Our laboratories first reported two novel classes of complex synthetic lipids, including alkylamidophosphocholines (PC lipid; CP-51) and alkylamidophosphate ester-linked lipid-AZT conjugates (lipid-AZT conjugates; CP-92), with selective and potent activity against human immunodeficiency virus type 1 (HIV-1). To extend these observations, we synthesized additional PC lipids and lipid-AZT conjugates (INK and INK-AZT conjugate) to evaluate their structure-activity relationships by testing for selectivity against infectious wild-type (wt) and drug-resistant HIV-1 replication, virus fusogenic activity and toxicity for mouse bone marrow cells. PC lipid compounds with medium chain lengths at positions 1 and 2 gave an improved selective index (SI). INK-3, with 12 and 8 carbons and INK-15, with 10 and 12 carbons were among the most selective when evaluated in CEM-SS cells. INK-14, a lipid-AZT conjugate where AZT replaced the choline in PC lipid INK-3, gave the highest SI of > 1250 against both infectious wt HIV-1 replication in CEM-SS cells and a clinical isolate in peripheral blood leukocytes. Notably, the PC lipid compounds INK-3 and INK-15, but not the lipid-AZT conjugate INK-14, were potent inhibitors of matched pairs of AZT-sensitive and AZT-resistant HIV-1 clinical isolates. INK-3 also inhibited replication of HIV-2 and TIBO-resistant HIV-1, and inhibited HIV-1-mediated fusogenic activity by 78, 41 and 9% in a dose-dependent manner. The TC50 for mouse bone marrow cells was > 100 micrograms/ml for INK-3 compared to 9.15-14.17 micrograms/ml for CP-51 and 0.142-0.259 microgram/ml for AZT. These data suggest that optimum PC lipid compounds are significantly less toxic than AZT and have high potential as novel therapeutic agents for AIDS.

Salmonellae activate tumor necrosis factor alpha production in a human promonocytic cell line via a released polypeptide.

Invasive strains of Salmonella spp. cause both systemic and localized infections in humans. The ability to resist infection and some aspects of the tissue pathology associated with the presence of Salmonella in the gastrointestinal tract have been shown to be mediated in part by the induction of tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine produced by activated macrophages and lymphocytes. Recent reports indicate that TNF-alpha is involved in the induction of human immunodeficiency virus replication by Salmonella in the latently infected human promonocytic cell line U1. In the present study, we investigated the effects of Salmonella on TNF-alpha production in U1 cells and a related cell line, U38. Unlike Escherichia coli or Yersinia enterocolitica, salmonellae rapidly induce TNF-alpha expression in these cells through a released factor(s). Time course experiments show that the kinetics of TNF-alpha production by U38 cells stimulated with Salmonella conditioned medium closely resemble those observed in response to live Salmonella. The observation that TNF-alpha levels are elevated by 60 min after exposure to either bacteria or their conditioned medium suggests that the soluble inducer is continuously released or shed by the bacteria and that the signal acts rapidly to increase TNF-alpha production. Furthermore, the ability to produce the TNF-alpha inducer is shared by at least four Salmonella serotypes and does not correlate with the abilities to invade and to survive within phagocytes. Treatment of active conditioned medium with trypsin, but not low pH, high temperature, or urea, significantly inhibits its TNF-alpha-inducing effect on U38 cells, a finding which points to a polypeptide product of Salmonella as the mediator of TNF-alpha production. Gel filtration chromatography of Salmonella conditioned medium reveals two peaks of activity, consistent with molecular masses of approximately 150 and 110 kDa.

Synthesis and evaluation of novel thymidine analogs as antitumor and antiviral agents.

Two series of thymidine analogs with a hydroxyalkylammonium(amine) moiety have been synthesized and evaluated for antitumor and antiviral activities. The hydroxyalkylammonium-(amine) group was introduced at the 5' position of the 2'-deoxyribose residue of thymidine or at a corresponding position in acyclic thymidine analogs. In order to increase the lipophilicity of these compounds and potentially enable them to cross the cell membrane, the free hydroxy group also was esterified with a long hydrocarbon chain. The hexadecanoyl analogs (compounds 1c, 1d, 7c, and 7d) showed moderate antitumor cytotoxicity against SV-28 and KB cell lines (IC50 approximately 20 microM). Compound 1d showed moderate anti-HIV activity (EC50 = 6.8 microM), while compound 5 showed weak anti-HIV activity (EC50 = 55 microM). None of the compounds showed antiherpes simplex virus activity.

Synthesis and antiproliferative and antiviral activity of 2'-deoxy-2'-fluoroarabinofuranosyl analogs of the nucleoside antibiotics toyocamycin and sangivamycin.

The glycosylation of 3,4-dicyano-2-[(ethoxymethylene)amino]pyrrole (7) with 2-deoxy-2-fluoro-alpha-D-erythro-pentofuranosyl bromide (2) furnished an anomeric mixture of nucleosides (8a,b). This mixture was separated, and the individual anomers were treated with methanolic ammonia to effect a concomitant deblocking and ring closure. This furnished both anomers of 2'-deoxy-2'-fluoro-ara-toyocamycin (9a,b). The cyano moiety of 9b was converted to the carboxamide moiety to furnish 2'-deoxy-2'-fluoro-ara-sangivamycin (10) and to the thiocarboxamide moiety to furnish 2'-deoxy-2'-fluoro-ara-thiosangivamycin (11). The target compounds 10 and 11 showed similar antiproliferative activity against L1210 cells in vitro, with IC50's of 3 and 5 microM. Antiviral evaluation revealed a somewhat different pattern of activity. All analogs, both alpha and beta anomers, were active against human cytomegalovirus (HCMV), albeit the beta anomers were most active. The beta anomers also were active against herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus (HIV). Compound 10 was most active in the series, ca. 10-fold more potent than 11; IC50's for 10 ranged from 4 to 25 nM for HCMV, HIV, and varicella zoster virus (VZV) and from 30 to 500 nM for HSV-1. Even though compound 10 was cytotoxic, which will probably preclude its use as an antiviral drug (IC50's = 0.2-5.5 microM), the difference between cytotoxicity and activity against HCMV, HIV, and VZV was sufficient to indicate specific activity against a viral target.

Membrane-interactive phospholipids inhibit HIV type 1-induced cell fusion and surface gp160/gp120 binding to monoclonal antibody.

Membrane-interactive phospholipids (PLs), previously evaluated for activity against HIV-1 in vitro, are known to affect late steps in viral replication. Studies were done to determine the effects of PL analogs on post-translational processing of HIV-1 proteins, binding of viral surface gp160/gp120 to CD4 receptor, and HIV-1-induced cell fusion. Results of this investigation indicated that PL alone (1-octadecanamido-2-ethoxypropyl-rac-3-phosphocholine, CP-51) and PL-AZT conjugate (1-octadecanamido-2-ethoxypropyl-rac-3-phospho-3'- azido-3'-deoxythymidine, CP-92) have no effect on HIV-1-induced syntheses or processing of gp160/gp120, pr51, p24, or p17 (including myristoylation) in infected cells. Progeny HIV-1 particles made in CP-92-treated H9IIIB cells contained gp120, pr51, and p24; however, these virus particles had reduced capacity to bind to CD4+ cells. Both CP-51 and CP-92 inhibited syncytium (cell fusion) formation between treated HIV-1-infected cells and uninfected CD4+ cells, and, they reduced HIV-1 gp160/gp120 binding to CD4+ cells and monoclonal antibody. These results suggest that anti-HIV-1 activity of PL compounds involves alteration of cell surface membranes and viral envelopes. Phospholipid compounds are a novel class of membrane interactive compounds with potential use in blocking the spread of HIV-1 infection and pathogenesis in AIDS.

Regulation of macrophage activation and human immunodeficiency virus production by invasive Salmonella strains.

Salmonellae possess the ability to adhere to and invade macrophages and in so doing trigger a number of intracellular events that are associated with cellular activation. As an initial approach to defining the mechanisms by which invasive salmonellae alter macrophage function, we have explored the impact of Salmonella infection on the production of human immunodeficiency virus (HIV) in U1 cells, a promonocytic cell line latently infected with the virus. Infection of U1 cells with a pathogenic strain of Salmonella enteritidis resulted in a marked induction of macrophage activation and HIV production. The stimulatory effect of salmonellae was mediated by signals other than lipopolysaccharide. Salmonella mutants with specific defects in invasion or intracellular survival were markedly less effective in the induction of HIV production. In contrast to S. enteritidis, strains of Yersinia enterocolitica, Legionella pneumophila, and Escherichia coli did not induce HIV production. However, all of these bacteria induced comparable levels of gene expression mediated by the HIV long terminal repeat. The results of this study are consistent with the notion that invasive salmonellae possess the ability to activate the macrophage by at least one mechanism that is not shared with several other species of gram-negative bacteria. Furthermore, the expression of this unique property is maximal with Salmonella strains that are not only invasive but also capable of prolonged survival within the macrophage. Our results indicate that the U1 cell line may be a very useful model system with which to examine the biochemical pathways by which internalized salmonellae modulate the activation state of the macrophage.

Activity of triciribine and triciribine-5'-monophosphate against human immunodeficiency virus types 1 and 2.

Triciribine (TCN) and its 5'-monophosphate (TCN-P) are novel tricyclic compounds with known antitumor activity; TCN-P is currently in phase II human clinical trials. We now report that these compounds have potent and selective activity against HIV-1 and HIV-2. Using a syncytial plaque assay, TCN and TCN-P were active against HIV-1 at 0.01-0.02 microM and had differential selectivities of 2250 and 1900, respectively, compared to 1850 for AZT. In contrast, TCN and TCN-P had minimal selectivity against human cytomegalovirus (50 and 27, respectively). TCN and TCN-P markedly inhibited HIV-1-induced p24 core antigen production, reverse transcriptase, and infectious virus production in a dose-dependent manner using HIV-1 acutely infected CEM-SS, H9, and persistently infected H9IIIB and U1 cells. In acutely infected PBL cells, TCN and TCN-P inhibited reverse transcriptase and infectious virus production but not p24 core antigen production. Using a microtiter XTT assay, TCN and TCN-P were active against a panel of HIV-1 and HIV-2 strains at IC50 values ranging from 0.02 to 0.46 microM. Evaluation of matched pairs of predrug and postdrug therapy HIV-1 isolates established that AZT-resistant and TIBO-resistant variants of HIV-1 were sensitive to TCN or TCN-P. Furthermore, unlike AZT and other fraudulent nucleosides, neither TCN, TCN-P, nor TCN-TP inhibited the viral reverse transcriptase. Thus, even though triciribine is a nucleoside chemically, it does not act biologically by classic nucleoside modalities but rather by a unique mechanism yet to be elucidated.

Pulmonary surfactant phospholipids modulate priming of rabbit alveolar macrophages for oxidative responses.

We investigated the effect of individual phospholipids contained in pulmonary surfactant (PS) on the macrophage-activating factor (MAF)-induced priming of rabbit alveolar macrophages (AMs) for oxidative responses elicited by phorbol myristate acetate (PMA) or opsonized zymosan (Op-Zym). AMs were incubated with MAF with or without phospholipids for 18 h. After incubation, oxidative responses were elicited with PMA (0.5 micrograms/ml) or Op-Zym (250 micrograms/ml) and monitored by chemiluminescence (CL) assays. The data indicate that natural surfactant inhibited MAF-induced priming of rabbit AMs for PMA- or Op-Zym-elicited oxidative responses. Artificial surfactant inhibited PMA-elicited CL responses but enhanced Op-Zym-elicited CL responses. Individual phospholipids differed in modulative activities. Dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylglycerol (DPPG), and phosphatidylinositol (PI) inhibited MAF-induced priming when the oxidative responses were elicited with PMA. Whereas DPPG inhibited Op-Zym-elicited oxidative responses, dipalmitoyl phosphatidylcholine (DPPC) and DOPC primed AMs for increased Op-Zym-elicited oxidative responses. DOPC did not affect the binding of phorbol dibutyrate to AMs, which suggests that reduced cell binding of phorbol ester was not responsible for the inhibition of PMA-elicited oxidative responses in AMs treated with DOPC. Similarly, DPPC, DOPC, and DPPG did not affect the number of zymosan particles phagocytosed by AMs compared to the control, which suggested that enhanced or reduced Op-Zym-elicited oxidative responses by phospholipids were not due to altered phagocytic activity of AMs. In conclusion, our data indicate that individual surfactant phospholipid differently modulates priming of AMs for oxidative responses, and the effect of individual phospholipids does not account for the effect of complete PS on priming of AMs.

Priming of rabbit alveolar macrophages for enhanced oxidative responses by herpes simplex virus type 2 infection.

The effect of herpes simplex virus type 2 (HSV-2) infection on the oxidative response in infant and adult rabbit alveolar macrophages (AM) was studied using either phorbol myristate acetate (0.5 microgram PMA/ml) or latex (250 micrograms/ml) as eliciting agents in a chemiluminescence (CL) assay. Results indicated that uninfected infant AM responded to a latex-elicited but not PMA-elicited CL response. HSV-2 infection (moi = 1.0) of infant AM for 2 hr at 37 degrees C did not alter the PMA or latex-elicited CL responses. In contrast, uninfected adult AM exhibited a markedly increased CL response when elicited with either PMA or latex. HSV-2 infection (moi = 1) of adult AM for 2 hr further increased both PMA- and latex-elicited CL responses. Increasing the moi to 10 inhibited both PMA- and latex-elicited CL responses. Incubation of uninfected control and HSV-2 infected adult AM for 18 hr at 37 degrees C resulted in spontaneous priming of the cells for increased CL responses. In the absence of PMA HSV-2 alone failed to elicit a CL response in adult AM. Infection with heat-inactivated HSV-2 (moi = 1.0 before heat inactivation) did not prime adult AM for enhanced CL responses. AM from BCG immunized adult rabbit produced a considerably higher level CL response that nonimmunized AM; however, HSV-2 infection of these cells did not further enhance the response. In summary, these data indicate that adult AM but not infant AM can be primed by active HSV-2 infection for an increased CL response elicited by either PMA or latex.

Synthesis and evaluation of novel ether lipid nucleoside conjugates for anti-HIV-1 activity.

Combinations of an amidoalkylphosphocholine, 8, and AZT have been found to cause an apparent synergistic action in suppressing infectious HIV-1 replication. In addition, amidoalkyl, oxyalkyl, and thioalkyl ether lipids have been chemically linked to anti-HIV-1 nucleosides (AZT and DDI) through phosphate and phosphonate linkages. These conjugates have shown promising in vitro anti-HIV-1 activity. Also, the conjugates have a 5-10-fold reduction in cell cytotoxicity compared to AZT alone. The most active compound, an amidoalkyl ether lipid-AZT conjugates, 4A, was found to have a differential selectivity of 1793 in a syncytial plaque assay. In comparison, AZT alone has a value of 1281.

Human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type 2 (HSV-2) can coinfect and simultaneously replicate in the same human CD4+ cell: effect of coinfection on infectious HSV-2 and HIV-1 replication.

Experiments were designed to determine whether HIV-1 and herpes simplex virus type 2 (HSV-2) coinfection leads to simultaneous replication of both viruses in the same human CD4+ cell (MT-4 cell line) and the possible effects of coinfection on infectious virus production. Results from transmission electron microscopy analysis revealed replication of typical HSV-2 nucleocapsids in the nucleus and budding of HIV-1 particles through the plasma membrane and through intracytoplasmic vacuoles containing enveloped HSV-2 particles in the same coinfected cell. Coinfection of HIV-1 persistently infected H9IIIB or promonocytic U1 cells with HSV-2 did not alter total production of infectious HSV-2 or the percentage of HSV-2 infectious centers compared with control H9 and U937 cells infected with HSV-2 alone. However, in coinfected promonocytic U1 cells HSV-2 induced infectious HIV-1 production measured by syncytial plaque assay. In summary, both HIV-1 and HSV-2 can coinfect and simultaneously replicate in the same human CD4+ cell. Interactions between HIV-1 and HSV-2 appear to be unidirectional, resulting in accelerated replication of HIV-1 as reported by Albrecht et al. (J Virol 1989;63:1861-1868), but not HSV-2 as shown by us.

Novel membrane-interactive ether lipid analogs that inhibit infectious HIV-1 production and induce defective virus formation.

A new class of membrane-active ether lipid (EL) analogs of platelet-activating factor were studied for in vitro anti-HIV-1 activity. Human T-cell (CEM-ss) monolayers or suspension cultures were used to determine effects of structural modifications of Type A phosphorus-containing and Type B nonphosphorus EL analogs on (a) the inhibitory concentration50 (IC50) for HIV-1 syncytial plaque formation and cell growth, and, (b) virus budding at the cell plasma membrane. Results indicate that representative Type A and Type B EL inhibit HIV-1 but not herpes simplex virus type 2 plaque formation when added before or up to 2 days after viral infection. Anti-HIV-1 activity does not involve direct inactivation of virus infectivity. Type A EL (IC50 range = 0.2-1.4 microM) with alkyoxy, alkylthio, or alkyamido substitution at glycerol position 1 and ethoxy or methoxy substitution at position 2, and Type B compounds (IC50 range = 0.33-0.63 microM) with an inverse choline or nitrogen heterocyclic substitution at position 3 have selective activity against HIV-1-infected T-cells. EL treatment of HIV-1-infected cells is associated with subsequent release of reverse transcriptase activity, but infectious virus production is inhibited with time after infection. Electron microscopic examination of HIV-1-infected and EL-treated cells revealed absence of detectable budding virus at the plasma membrane but presence of intracytoplasmic vacuolar virus particles. In summary, these data suggest that EL analogs are a novel class of agents that induce defective intracytoplasmic vacuolar HIV-1 formation in T-cells. Being membrane interactive, EL are ideally suited for combination chemotherapy with DNA-interactive anti-HIV nucleoside analogs.

Prenatal detection of non-cardiac rhabdomyosarcoma.

The most prevalent soft tissue tumour in children is rhabdomyosarcoma. These tumours may develop within or outside of muscle anywhere in the body and at any age. We report what is apparently the earliest case of non-cardiac rhabdomyosarcoma detected prenatally.